Construction of a novel recombinant vector carrying

زمینه و هدف: ‌لیشمانیوز به گروهی از بیماریهای انگلی ناشی از گونه های مختلف لیشمانیا اطلاق می شود که اشکال بالینی گوناگونی دارد. این انگل در انسان و حدود 100 گونه حیوان ایجاد بیماری می نماید و در بخش های وسیعی از جهان و از جمله ایران شایع است. یکی از زمینه های دستیابی به واکسنی مؤثر علیه لیشمانیا، دستکاری ژنتیکی این تک یاخته و استفاده از انگل مهندسی شده به عنوان واکسن می باشد. هدف از این تحقیق طراحی و ساخت کاست ژنی جدیدی است که به منظور وارد ساختن ژنهای خودکشی سلولی شامل HSV-tk و Yeast-cd به ژنوم لیشمانیا طراحی گردیده است. روش بررسی: در یک مطالعه آزمایشگاهی ابتدا دو قطعه ژن تیمیدین کیناز ویروس هرپس سیمپلکس (HSV-tk) و سیتوزین دآمیناز مخمر ساکارومایسس سرویزیه (Yeast-cd) به همراه ژن آلفا توبولین لیشمانیا (atub) با ترتیب tk-αtub-cd در پلاسمید pBluscript کلون شدند. سپس مجموعه ژنی مذکور از پلاسمید pBluscript خارج گردیده و در پلاسمید pF4X1.4sat کلون گردید و به منظور تأیید کانستراکت نهایی از هضم برش توسط آنزیم های محدود الاثر استفاده شد. یافته ها: قطعات ژنی tk-αtub-cd با موفقیت در پلاسمید pBluscript کلون گردیده و صحت کلونینگ مورد تأیید قرار گرفت. سپس این مجموعه ژنی در پلاسمید pF4X1.4sat درج شد و صحت حضور و ترتیب قرارگیری این ژنها اثبات گردید. نتیجه گیری: سیستم طراحی شده در این تحقیق می تواند دو ژن خودکشی سلولی را به درون ژنوم لیشمانیا منتقل کند و در تحقیقات آینده در زمینه دستیابی به واکسن زنده علیه لیشمانیا مورد استفاده قرار گیرد.

High Frequency of Class 2 and 3 Integrons Related to Drug-Resistance in Clinical Isolates of Diarrheagenic E. coli in Iran

Background: Integrons are mobile genetic elements able to obtain the antibiotic resistance gene cassettes. The prevalence of integrons in the Enterobacteriaceae family has been varied and played an important role in the development of the drug resistant bacteria. The present study aimed to investigate the contribution of class 2 and 3 integrons in drug resistant Diarrheagenic Escherichia coli strains.Materials and Methods: The 164 Diarrheagenic E. coli collected from feces samples of children in the Yasuj –Iran and all isolates were identified by standard biochemical tests. The antimicrobial susceptibility for 14 antibiotics, which are used conventionally was determined by disk diffusion. The presence of class 2 and 3 integrons in all isolates was investigated by PCR.Results: Of 164 E. coli isolates from children, 80.49% carried class 2 integron and the length of the amplicons ranged from 800 bp to 2 kb. Class 3 integrons were identified among 24 E. coli isolates. All the E.coli isolates were susceptible to imipenem and the greatest resistance was correspondent to nalidixic acid. A significant correlation was revealed between Class 2 integron and resistance to kanamycin, amikacin, gentamicin, ceftazidime, chloramphenicol and cephalexin. The presence of class 3 integron was significantly associated with resistance to ampicillin, gentamicin, streptomycin, kanamycin, tetracycline and trimetoprime-sulfametoxazol.Conclusion: The results indicated that integrons are widespread in Diarrheagenic E. coli and its carriage contributed significantly to the emergence of resistance among Diarrheagenic E. coli. However, factors leading to the wide spread of integrons are still to be determined.

Prevalence of aadA1, aadA2, aadB, strA and strB genes and their associations with multidrug resistance phenotype in Salmonella Typhimurium isolated from poultry carcasses

This study aimed to assess the prevalence of aminoglycoside resistance genes in S. Typhimurium isolated from poultry carcasses in Iran, and to reveal the most prevalent patterns of antimicrobial resistance. A total number of 300 samples of poultry carcasses were analyzed. Salmonella was isolated from 245 samples (81.66%). Multiplex PCR showed that 56.3% of the samples belonged to serovar S. Typhimurium and the remainder (43.6%) contained the rest of serovars. The highest rate of drug resistance was observed for tetracycline (97.0%), nalidixic acid (87.0%) and amoxicillinclavulanic acid (67.4%). These serovars, however, were sensitive to cefotaxime (84.8%), sulfamethoxazole trimethoprim (77.6%) and gentamicin (71.0%). aadA1 gene was detected in 63 isolates (45.6%), aadA2 in 48 isolates (34.7%), aadB in 43 isolates (31.1%), strA in 52 isolates (37.6%) and strB in 31 isolates (22.4%). High prevalence of aminoglycoside resistance genes in S. Typhimurium was shown. Furthermore, there was a significant association (P < 0.02) between the presence of aadA1, aadA2, strA and strB genes and resistance to streptomycin. Also, there was a significant association (P < 0.001) between the presence of aadB gene and resistance to kanamycin and gentamicin

Isolation of Escherichia coli O157: H7 in sheep meats using cultural and PCR method

زمینه و هدف: اشرشیاکلی O157:H7 به عنوان یک عامل اسهال، کولیت خونریزی‌دهنده و سندرم اورمی همولیتیک در سراسر جهان شناخته شده است. گوشت آلوده به مدفوع حیوانی احتمالاً منبع اصلی عفونت اشرشیاکلی O157:H7 می‌باشد. این مطالعه با هدف بررسی میزان شیوع اشرشیاکلی O157:H7 در نمونه‌های گوشت گوسفند در اصفهان با استفاده از روش کشت و واکنش زنجیره ای پلی مراز (PCR) انجام گرفت. روش بررسی: در این مطالعه توصیفی - تحلیلی 148 نمونه گوشت گوسفند کشتار شده در کشتارگاه اصفهان از مرداد 1386 تا 1388 به صورت تصادفی جمع آوری شدند. نمونه ها در آبگوشت تریپتون سوی حاوی مکمل نووبیوسین (TSB-n) به عنوان یک محیط غنی کننده و سپس محیط مک کانکی آگار سوربیتول دار حاوی مکمل سفکسیم و تلئوریت پتاسیم (CT-SMAC) به عنوان یک محیط انتخابی کشت داده شد. کلنی های مشکوک اشرشیاکلی O157:H7 جدا شده از روش های باکتریولوژی به وسیله آزمون زنجیره‌ای پلیمراز مورد ارزیابی قرار گرفت. یافته ها: بر اساس آزمون کشت به ترتیب 43 (1/29) و 10(8/6) نمونه از نظر اشریشیاکلی و اشرشیاکلی O157:H7 مثبت بود. اما تنها 5 نمونه از اشرشیاکلی‌های سوربیتول منفی در آزمون واکنش زنجیره‌ای پلیمراز به عنوان اشرشیاکلی O157:H7 تشخیص داده شدند. شیوع فصلی اشرشیاکلی O157:H7 در نمونه‌های بررسی شده بین0 تا 7/9 بود و بالاترین میزان آن در فصول بهار و تابستان مشاهده شد (05/0

Prevalence and molecular characterization of rotaviruses as causes of nosocomial diarrhea in children

Rotaviruses have been confirmed as causative agents of nosocomial gastroenteritis in children, but limited data exist concerning the epidemiology of nosocomial rotavirus gastroenteroentritis in Iran. The aim of this study was to determine the prevalence and molecular characteristics of rotavirus in children less than five years old with nosocomial diarrhea in Shahrekord (southwest of Iran). This cross-sectional study was conducted between December 2010 and October 2011. The study population consisted of children aged 6 to 60 months who were hospitalized in the pediatric ward of Hajar Hospital in Shahrekord, Iran, due to diseases other than diarrhea. Nosocomial diarrhea was defined as that occurring more than 48 hours after admission to the hospital for non-diarrheal causes. Rotavirus and G genotypes were determined by seminested reverse transcriptase polymerase chain reaction in 100 stool samples. In these 100 samples, the prevalence of rotavirus infection was 30%; the most common genotyes were G1 (20%) and G9 (20%). According to the findings of the study, genotyping of rotavirus is necessary to monitor changes in strain prevalence. Identifying strains over time could affect future vaccine strategies and detect any regional differences of genotype prevalence

Prevalence of Nosocomial Diarrhea Due to Adenoviruses 40 and 41 in a Paediatric Ward in Iran.

BACKGROUND Enteric adenoviruses 40 (Ad40) and adenovirus 41 (Ad41) have been shown to be a significant cause of paediatric gastroenteritis worldwide, but no data are available for nosocomial diarrhea due to adenovirus in Iran. AIM The present study was performed to determine the incidence of Ad40 and Ad41 in children less than five years with nosocomial diarrhea in Shahrekord, southwest Iran. MATERIALS AND METHODS Adenovirus was detected by polymerase chain reaction (PCR) in stool samples collected during one year (2010-2011) from children less than five years with nosocomial diarrhea admitted to a paediatric center in Shahrekord, Iran. Nosocomial diarrhea was defined as those occurring more than 72 hours after admission to hospital for non-diarrheal causes. PCR technique was used for investigation of Ad40 and Ad41. RESULTS In total of 100 samples, Ad40 and Ad41 DNA was found to be positive in 14/100 (14%), and 8/100 (8%) of diarrheic patients less than five years, respectively. CONCLUSION Ad40 and Ad41 are important causes of nosocomial diarrhea in less than five-year, hospitalized Iranian children

Antibiotic resistance and distribution of beta-lactamase resistance genes in Escherichia coli strains isolated from urinary tract infection in women and children in the city Farsan

زمینه و هدف: عفونت دستگاه ادراری از شایع ترین عفونت&zwnj;ها در زنان و کودکان است که در این میان باکتری اشریشیاکلی عامل اصلی عفونت بشمار می رود. مقاومت علیه آنتی بیوتیک&zwnj;های مورد استفاده، مشکل مهمی در پروسه درمانی می باشد. این مطالعه به منظور تعیین مقاومت آنتی بیوتیکی و بررسی شیوع ژنهای مقاومت نسبت به آنتی بیوتیک ها می باشد. روش بررسی: مطالعه توصیفی-مقطعی از تعداد 95 بیمار زن و کودک مبتلا به عفونت ادراری مراجعه کننده به کلینیک شهرستان فارسان در سال 1392 انجام شده است. نمونه های باکتری پس از رشد در محیط بلاد آگار و EMB با استفاده از روش آنتی بیوگرام با روش Kirby-bauer و هاله عدم رشد مورد بررسی قرار گرفتند. سپس جهت تعیین شیوع ژنهای مقاومت CTX، CTX-M، TEM و B-SHV از روش PCR استفاده گردید. یافته ها: از تعداد 95 نمونه اشرشیاکلی بیشترین مقاومت به ترتیب به آمپی سیلین(78/95)، تری متوپریم-سولفامتوکسازول(10/62)، نیتروفورانتوئین(10/42) و بیش ترین حساسیت مربوط به آنتی بیوتیک های سیپروفلوکساسین(31/46) و سفتریاکسون(21/44) گزارش گردید. همچنین نتایج PCR نشان داد CTX بیشترین و SHV-B کمترین شیوع در بین ژن&zwnj;های مقاومت را دارا می باشند. نتیجه گیری: نتایج این مطالعه نشان داد شیوع باکتری اشرشیاکلی تولید کننده ژن های مقاومت به بتالاکتاماز در حال افزایش است. همچنین در درمان اولیه عفونت های ادراری بهتر است از آنتی بیوتیک های آمپی سیلین، تری متوپریم-سولفامتوکسازول و نیتروفورانتوئین کمتر استفاده گردد. کلمات کلیدی: عفونت های ادراری، اشرشیاکلی، CTX-M، مقاومت، PC

Presence of Listeria monocytogenes in silage products of Shahrekord city

Objective: To investigate the presence of Listeria monocytogenes in the silage samples. Methods: Silage samples obtained from 150 different farms in Shahrekord city (Iran) and after DNA extraction, all samples were analyzed by PCR technique using one pair of primers for presence of this pathogen. The amplified products were detected on 1.5% agarose gel electrophoresis. Results: Listeria monocytogenes was isolated in 4 (2%) of the 150 samples. The detection of this bacterium from silage samples in Shahrekord city indicated that these products could create a serious risk in public health of animal and human. The findings showed that in positive silage samples for Listeria monocytogenes, the pH value was about five and it was due to bacterial activity in these products. Conclusions: The quality of silage and hygiene parameters and good herd health management play an important role in the microbiological quality of herd and farm. Considering the high specificity and sensitivity of the employed PCR technique, it is recommended to be useful technique for identification of Listeria monocytogenes

GENERATION OF DIVALENT DNA VACCINE BASED ON p39 AND shiga-like toxin 2 (stx2) GENES

The virulence factors such as shiga-like toxin (Stx) and immunogenic P39 protein in Escherichia coli and Brucella melitensis are related to disease of digestive system in human worldwide. In the present study the stx2 and p39 genes were cloned into expression plasmid pEEF1D-FLAG (pcDNA 3.1(+)) as a divalent DNA vaccine candidate. The Enterohemorrhagic E. coli ATCC 3081 and smooth virulent B. melitensis strain M5 were obtained and cultured on specific media. Bacterial DNA was extracted from colonies and was used for p39 and stx2 genes amplification by PCR. The amplified products on 2% agarose gel electrophoresis were revealed 285 and 1220 bp fragments for stx2 and p39 genes, respectively. Each amplified genes were T/A cloned into pGEMT easy vector and pGEM-T-stx2 and pGEM-T-p39 were produced. The stx2 and p39 genes were sub-cloned in linearized expression vector (pcDNA 3.1(+)) using HindIII, XhoI and XbaI restriction enzymes and pCDNA3-stx2-p39 was generated. This final construct was confirmed by PCR and enzymes digestion. The results were showed stx2 and p39 genes were sub-cloned, successfully into pcDNA 3.1(+) to generate pcDNA 3.1(+)-stx2-p39 recombinant vector. According to these findings novel recombinant pcDNA 3.1(+)-stx2-p39 construct that was produced in this study could be useful as DNA vaccine candidate in animal models against shiga-like toxin producing E. coli and virulence B. melitensis strains in future studies

The effects of pBudCE4.1-azurin-MAM-A recombinant vector on IL-2, IL-6, IL-7, and IL-10 expressions in laboratory mice

Background and aims: Breast cancer is one of the most common types of malignancy in women with morbidity and mortality (15.0%) in the world. The antitumor activity of azurin protein produced by Pseudomonas aeruginosa has been described before. Mammaglobin-A (MAM-A) protein is especially expressed in 40%-80% of breast cancer types and this protein is a very specific molecular marker for stimulating the immune system. Accordingly, this study investigated the effects of pBudCE4.1-azurin-MAM-A recombinant vector on the induction of the immune system in laboratory mice by the real-time polymerase chain reaction (PCR) method. Methods: The pBudCE4.1-azurin-MAM-A recombinant and empty vectors were purchased and then separately transformed into Escherichia coli for multiplying. Next, each plasmid was extracted and the accuracy of transformation was confirmed by the PCR. These recombinant and empty (control) vectors were separately infused into the thigh muscle of the animals and the healthy group was infused with phosphatebuffered saline. The infusion sites, blood specimens, as well as the serum of the animals were collected and examined by serological and molecular tests. Results: Molecular and serological studies showed that the serum and expression levels of IL-2, IL-6, IL-7, and IL-10 in infused mice with pBudCE4.1-azurin-MAM-A recombinant vector significantly increased compared to healthy animals and injected mice with an empty vector (P<0.05). Conclusion: In general, the findings revealed that the pBudCE4.1-azurin-MAM-A recombinant vector can stimulate the immune system of the mouse by an increase in the expression levels of IL-2, IL-6, IL-7, and IL-10. Thus, it would be better to examine the effects of this recombinant vector as a DNA vaccine on the prevention and treatment of breast cancer. Keywords: Azurin, MAM-A, Recombinant vector, Breast cance